Development of models to improve vascularisation within islet-laden constructs with 3D bioprinting

Conventional pancreatic islet transplantation is a promising life-changing treatment for individuals with severe type 1 diabetes (T1D). However, due to the nature of islet isolation and purification, there is a complete disruption of the intra-islet vasculature system resulting in islet death and reduced function. Post-transplantation, the rate of intra-islet revascularisation and vascular density will determine the survival rate and functional capacity of these transplanted islets. In attempts to improve vascularisation in islets, three dimensional (3D) bioprinting is a popular method to biofabricate large tissue constructs that mimic the natural organisation found in the body. Different 3D bioprinting methods are employed to promote vascularisation via utilisation of different biomaterials and cell types. However, the quest is still ongoing to create vascularisation in a timely manner at early stages of islet transplantation. Although various 3D bioprinting techniques have been developed to support islet viability and function, the challenge of effective vascularisation between islet-laden construct and the host’s tissue remains.

Therefore, this PhD project investigates a novel biofabrication approach to enhance both the rate of intra-islet revascularisation and density through creating vascularisation in islet-laden constructs. Our alternative approach utilises the Janus nozzle to create colinear constructs. We hypothesised that the creation of void spaces via the use of a sacrificial bioink can support oxygen and nutrient exchange without hindrance from the bioinks. Alongside this, empty space in the 3D construct can potentially enable the endothelial cells to form inter-connected vessel like formations between neighbouring cells further supporting the creation of vascularisation.

In Chapter 2, bioink optimisation of an alginate/GelMA/gelatin of two bioink blends was developed to enable bioprinting at room temperature to remove the need for external temperature control. To evaluate the capabilities of the bioink blend, the first 3D bioprinting method explored was coaxial bioprinting. Filament extrusion and bioprinting of a scaffold were conducted to evaluate the printability but also the structural integrity of the bioink compositions. To further determine the characteristics of the bioink blend, rheology and mechanical characterisation was conducted to understand the behaviour of the bioink blend. Through these testings, the bioink showed shear thinning properties and self-recovering properties to support the 3D bioprinting extrusion process. Alongside this, mechanical strength of the scaffolds was supported by the Young’s modulus. Exposure of the bioink to human umbilical vein endothelial cells (HUVECs) showed cellular viability.

In Chapter 3, an alternative method was explored utilising the Janus nozzle to contrast a colinear construct, where two bioinks are simultaneously side-by-side. This development of the model supports the concept of creating empty spaces, voids, in the 3D environment through the incorporation of a sacrificial bioink. Further optimisation of the bioinks was conducted to support this alternative model through the addition of alginate methacrylate (AlgMA) to support structural integrity. Inclusion of AlgMA showed stability at 37°C as compared to the model in Chapter 2. Moving ahead, stability at body temperature indicates the potential for transplantation post- 3D bioprinting. Additionally, HUVEC function and viability was supported in this colinear model with interconnected vessel-like formations forming in a 24-hour period post 3D bioprinting. Vessel maturation was observed between day 1 and day 7 through live/dead confocal staining showing the HUVECs were supported in this environment.

Continuing in Chapter 4, in vivo transplantation of the colinear cell-laden constructs with islets showed the potential of angiogenesis in the subcutaneous region of mice. The 3D bioprinted cell-laden constructs can be manipulated to be placed in the subcutaneous region and remain intact by day 14 and day 21 post-transplantation. Presence of the human islets in the graft and their hormone insulin can confirm the presence of the hormones in the islets. Histological analysis can identify the construct at the interface of the subcutaneous region. Visualisation of blood vessels can be observed upon extraction of the graft. The colinear model holds promising results for the creation vascularisation to improve outcome of islets post-transplantation.

Finally in Chapter 5, an economic analysis was conducted to determine whether implementation of 3D bioprinting as an alternative treatment to conventional islet transplantation can be cost-effective. The report has been based on assumptions and shows that 3D bioprinting can significantly reduce the economic burden of the islet transplantation procedure. The evidence in this report provides a foundation as to why implementing a new method can reduce economic burden and increase quality of life.

These results show the potential of 3D bioprinting as an alternative method to break down the barrier by tackling vascularisation to enable broader application of islet transplantation.